An Innovative Aggregation-Induced Emission-Based NIR Fluorescent Probe for Visualizing Carboxylesterases in Living Cells, Zebrafish, and Tumor-Bearing Mice

In the human body, carboxylesterases (CEs) play crucial roles in xenobiotic metabolism and lipid homeostasis. But abnormal expression of CEs is highly associated with some diseases, such as hyperlipidemia, diabetes, and liver cancer. Therefore, it is of great importance to develop an efficient tool for the accurate detection of CEs in living organisms. Herein, an innovative near-infrared (NIR) fluorescent probe, TTAP−AB, was designed for CE detection based on the aggregation-induced emission (AIE) mechanism. This probe exhibits rapid response (2 min), excellent sensitivity (limit of detection = 8.14 × 10−6 U/mL), and high selectivity to CEs. Additionally, owing to its good biocompatibility, the TTAP−AB probe enables the monitoring of dynamic changes in CE levels under drug-induced modulation in living cells and zebrafish. More importantly, the TTAP−AB probe was successfully employed to image liver tumors and assist in tumor resection through the real-time monitoring of CEs, indicating that TTAP−AB is promising to guide liver cancer surgery. Therefore, the TTAP−AB probe can not only enrich the strategies for CE detection in biological systems but also has great potential for some clinical imaging applications, including medical diagnosis, preclinical research, and imaging-guided surgery.


Introduction
Carboxylesterases (CEs, with EC 3.1.1.1),as essential members of the serine hydrolase superfamily, are found in various tissues of the human body, especially the liver and intestine [1].CEs play key roles in catalyzing the hydrolysis of endogenous esters, thioesters, carbonates, carbamates, and amides.In addition, they are involved in the metabolic elimination of various xenobiotics, including ester prodrugs, pesticides, and environmental toxicants [2].Apart from metabolizing various exogenous and endogenous substances, CEs also have vital physiological functions in lipid homeostasis that convert monoacylglycerides into free fatty acids [3].However, abnormal CE expression is tightly correlated with many diseases, such as hyperlipidemia, diabetes, atherosclerosis, and even liver cancer [4,5].Therefore, exploring an effective strategy to track the distribution of CEs and evaluate their activity variation in cells or tissues is of great significance for both clinical diagnosis and the treatment of various diseases.
Aggregation-induced emission (AIE), a unique optical phenomenon whereby a class of luminogens were nearly non-emissive in a dilute solution but highly emissive in the aggregated state, was first coined by Tang et al. in 2001 [25].In this regard, turn-on bioprobes could be easily constructed by taking advantage of the AIE effect.It was believed that the nanoaggregates of the AIE-based probes would possess better photostability and higher signal reliability than the single molecule of conventional probes.Furthermore, the negligible background noise of AIE-based probes rendered them especially attractive for the continuous monitoring of biological processes without repeated washing steps [26].On account of the above advantages, AIE-based probes have been widely used in bioimaging and biological testing [27].But, as far as we know, the AIE-based fluorescent probes for CE detection have been rarely reported.Dai and co-workers reported an AIE-based fluorescent probe for monitoring CEs in HepG2 cells, but the fluorescence signal was in the visible region (λ em = 589 nm) [22]; therefore, this probe was unsuitable for the fluorescence detection of CEs in vivo.
Herein, we developed a novel NIR fluorescence TTAP−AB probe for detecting CEs with the AIE mechanism (Scheme 1).The TTAP−AB probe was designed based on a typical D-π-A structure, in which the conjugated C=C double bond bridged the triphenylamine-thiophene (TT) skeleton (electron donor, D) and the acrylonitrilepyridinium (AP) moiety (electron acceptor, A).Owing to the cationic pyridinium moiety, the TTAP−AB probe was well dissolved in the PBS buffer.As the presence of CEs increased, the acetoxy-benzyl recognition group was hydrolyzed and broken by CEs, which triggered the self-elimination reaction and finally released the AIE-active fluorophore TTAP.Because TTAP was not dissolved in the PBS buffer, its aggregation aroused an intense NIR emission (λ em = 692 nm).In addition, the TTAP−AB probe exhibited a high selectivity, fast response (within 2 min), and low limit of detection (LOD: 8.14 × 10 −6 U/mL) for CEs.Owing to its good biocompatibility and excellent cell-tissue penetrability, TTAP−AB could monitor the dynamic change in CE levels induced by 5-fluorouracil (an anti-tumor drug) and CE inhibitors in living cells and zebrafish.More importantly, TTAP−AB was successfully employed to image the liver tumor and assist tumor resection through the real-time monitoring of CEs, indicating that the TTAP−AB probe was promising to guide liver cancer surgery.

Synthesis and Characterization of the TTAP−AB Probe
As shown in Scheme 2, the TTAP−AB probe was synthesized via a three-step reaction.Firstly, the Wittig reaction of compound 1 and (1,3-dioxolan-2-ylmethyl)triphenylphosphonium bromide produced intermediate 2. Subsequently, a Knoevenagel condensation reaction was performed using compound 2 and 2-(pyridin-4-yl)acetonitrile to produce fluorophore TTAP.Finally, the subsequent quaternization reaction of TTAP with 4-(bromomethyl)phenyl acetate produced the TTAP−AB probe with a 62% yield.The structures of the above compounds were confirmed using 1 H NMR, 13 C NMR, and HRMS, as shown in the Supporting Information (Figures S8−S15).

Spectroscopic Response of TTAP−AB to CEs
First, the absorption and fluorescence spectra of the pure TTAP−AB probe were investigated in the absence and presence of CEs; as shown in Figure 1a, TTAP−AB (5 µM) showed an absorption peak at 548 nm and negligible fluorescence in the PBS buffer.Upon treatment with CEs, a new absorption peak was observed at 480 nm, accompanied by a change in color from purple to orange, which may have been caused by a hydrolytic reaction.According to the formula "A = εbc", the absorption coefficients of TTAP−AB before and after treatment with CEs were calculated to be 4.17 × 10 4 L•mol⁻¹•cm⁻¹ and 1.10 × 10 5 L•mol⁻¹•cm⁻¹, respectively (Figure S1).Meanwhile, the fluorescence intensity at 692 nm Scheme 1.The proposed mechanism of the TTAP−AB probe to detect CEs.

Synthesis and Characterization of the TTAP−AB Probe
As shown in Scheme 2, the TTAP−AB probe was synthesized via a three-step reaction.Firstly, the Wittig reaction of compound 1 and (1,3-dioxolan-2-ylmethyl)triphenylphosphonium bromide produced intermediate 2. Subsequently, a Knoevenagel condensation reaction was performed using compound 2 and 2-(pyridin-4-yl)acetonitrile to produce fluorophore TTAP.Finally, the subsequent quaternization reaction of TTAP with 4-(bromomethyl)phenyl acetate produced the TTAP−AB probe with a 62% yield.The structures of the above compounds were confirmed using 1 H NMR, 13 C NMR, and HRMS, as shown in the Supporting Information (Figures S8-S15).

Synthesis and Characterization of the TTAP−AB Probe
As shown in Scheme 2, the TTAP−AB probe was synthesized via a three-step reaction.Firstly, the Wittig reaction of compound 1 and (1,3-dioxolan-2-ylmethyl)triphenylphosphonium bromide produced intermediate 2. Subsequently, a Knoevenagel condensation reaction was performed using compound 2 and 2-(pyridin-4-yl)acetonitrile to produce fluorophore TTAP.Finally, the subsequent quaternization reaction of TTAP with 4-(bromomethyl)phenyl acetate produced the TTAP−AB probe with a 62% yield.The structures of the above compounds were confirmed using 1 H NMR, 13 C NMR, and HRMS, as shown in the Supporting Information (Figures S8−S15).

Spectroscopic Response of TTAP−AB to CEs
First, the absorption and fluorescence spectra of the pure TTAP−AB probe were investigated in the absence and presence of CEs; as shown in Figure 1a, TTAP−AB (5 µM) showed an absorption peak at 548 nm and negligible fluorescence in the PBS buffer.Upon treatment with CEs, a new absorption peak was observed at 480 nm, accompanied by a change in color from purple to orange, which may have been caused by a hydrolytic reaction.According to the formula "A = εbc", the absorption coefficients of TTAP−AB before and after treatment with CEs were calculated to be 4.17 × 10 4 L•mol⁻¹•cm⁻¹ and 1.10 × 10 5 L•mol⁻¹•cm⁻¹, respectively (Figure S1).Meanwhile, the fluorescence intensity at 692 nm Scheme 2. The synthetic route of the TTAP−AB probe.

Spectroscopic Response of TTAP−AB to CEs
First, the absorption and fluorescence spectra of the pure TTAP−AB probe were investigated in the absence and presence of CEs; as shown in Figure 1a, TTAP−AB (5 µM) showed an absorption peak at 548 nm and negligible fluorescence in the PBS buffer.Upon treatment with CEs, a new absorption peak was observed at 480 nm, accompanied by a change in color from purple to orange, which may have been caused by a hydrolytic reaction.According to the formula "A = εbc", the absorption coefficients of TTAP−AB before and after treatment with CEs were calculated to be 4.17 × 10 4 L•mol −1 •cm −1 and 1.10 × 10 5 L•mol −1 •cm −1 , respectively (Figure S1).Meanwhile, the fluorescence intensity at 692 nm (F 692 ) was enhanced continuously upon a gradual increase in CE concentration (0.00-0.25 U/mL) (Figure 1b).Notably, the F 692 values exhibited a good linear relationship (R 2 = 0.9978), with CE concentrations ranging from 0.00 to 0.18 U/mL (Figure 1c).Based on the formula 3σ/k, the limit of detection (LOD) was calculated to be 8.14 × 10 −6 U/mL (Section S2), implying a much higher sensitivity than many other CE probes [10,11,[13][14][15]19,21,22].Moreover, as shown by the dynamic light scattering (DLS) data, an initial signal peak was not found, indicating that the TTAP−AB probe dissolved and dispersed well in the PBS buffer.However, after treatment with 0.25 U/mL of CEs, the average size of the formed nanoparticles was around 240 nm (Figure 1d).It is clear that the CEs caused the aqueous TTAP−AB solution to form nanoaggregates, consequently resulting in bright NIR emissions.
Molecules 2024, 29, x FOR PEER REVIEW 4 of 15 (F692) was enhanced continuously upon a gradual increase in CE concentration (0.00-0.25 U/mL) (Figure 1b).Notably, the F692 values exhibited a good linear relationship (R 2 = 0.9978), with CE concentrations ranging from 0.00 to 0.18 U/mL (Figure 1c).Based on the formula 3σ/k, the limit of detection (LOD) was calculated to be 8.14 × 10 −6 U/mL (Section S2), implying a much higher sensitivity than many other CE probes [10,11,[13][14][15]19,21,22].Moreover, as shown by the dynamic light scattering (DLS) data, an initial signal peak was not found, indicating that the TTAP−AB probe dissolved and dispersed well in the PBS buffer.However, after treatment with 0.25 U/mL of CEs, the average size of the formed nanoparticles was around 240 nm (Figure 1d).It is clear that the CEs caused the aqueous TTAP−AB solution to form nanoaggregates, consequently resulting in bright NIR emissions.

The Studies of Selectivity, Response Time, and pH Effect
To evaluate its selectivity toward CEs, the TTAP−AB probe was incubated with various potential interferents, including 0.60 U/mL of a common enzyme (acetyl cholinesterase (AchE), carbonic anhydrase I (CAI), xanthine oxidase (XO), peroxidase (POD), carboxypeptidase A (CPA), and leucine aminopeptidase (LAP)); 100 µM each of amino acids (glutamic acid (Glu), cysteine (Cys), glutathione (GSH), and homocysteine (Hcy)); and 100 µM each of common ions (K + , Na + , Zn 2+ , Cu 2+ , Mg 2+ , Cl − , CO 3 2− , SO 3 2− , S 2− , and H 2 PO 4 − ).The fluorescence of TTAP−AB at 692 nm was only marginally triggered by CEs (Figure 2a), and the quantum yield of TTAP−AB+CEs (Φ = 0.384) was almost 15-fold higher than that of TTAP−AB (Φ = 0.025) (Section S3); conversely, the other species caused negligible fluorescence changes.To further investigate the CEs-dependent selective response, an anti-interference test was carried out.As shown in Figure 2b, even when coexisting with different interferents, CEs could still significantly enhance the fluorescence intensity of TTAP−AB.The carboxyl groups of Cys and GSH led to the protonation of pyridine moieties in TTAP, which improved the water solubility of TTAP and reduced its aggregation in the PBS buffer, consequently causing a slight decrease in fluorescence intensity (Figure 2b).In summary, these results suggest that the TTAP−AB probe possesses a relatively high selectivity for CEs compared to other interferential species, setting a solid foundation for CE detection in subsequent biological experiments.
when treated with CEs.As displayed in Figure 2d, the fluorescence intensity of TTAP−AB (5 µM) was basically not affected at different pHs, indicating that TTAP−AB displayed excellent pH stability.After treatment with CEs (0.20 U/mL), the fluorescence intensity at 692 nm was significantly increased when the pH was 6.0-9.0.In a strongly acidic medium (pH ≤ 5), the TTAP produced from TTAP−AB treated with CEs was dissolved in an aqueous solution due to protonation and thus was unable to emit in the NIR, while in strong alkaline conditions (pH ≥ 10), the reaction of CEs toward TTAP−AB was reduced [15,18], which hampered the release of fluorophore TTAP and prevented the generation of NIR fluorescence.Despite this, the TTAP−AB probe was still able to monitor CEs in physiological environments.Previous reports show that most fluorescent probes require a long response time (over 10 min) for CEs [10][11][12][13][14][15][17][18][19][20][21][22], making them unsuitable for real-time analysis in living organisms.In view of this, we studied the time-dependent fluorescence intensity response of the TTAP−AB probe to CEs treatment.When TTAP−AB (5 µM) was excited at 505 nm, its fluorescence intensity remained unchanged for 30 min, indicating that it possessed excellent photostability.By contrast, when TTAP−AB was treated with CEs (0.20 U/mL), the fluorescence intensity at 692 nm increased rapidly and reached a plateau within 2 min (Figure 2c).It is clear that TTAP−AB exhibited a much faster response to CEs than most reported probes (Table S1).For broader biological applications, we investigated the impacts of environmental pH on the fluorescence response of the TTAP−AB probe when treated with CEs.As displayed in Figure 2d, the fluorescence intensity of TTAP−AB (5 µM) was basically not affected at different pHs, indicating that TTAP−AB displayed excellent pH stability.After treatment with CEs (0.20 U/mL), the fluorescence intensity at 692 nm was significantly increased when the pH was 6.0-9.0.In a strongly acidic medium (pH ≤ 5), the TTAP produced from TTAP−AB treated with CEs was dissolved in an aqueous solution due to protonation and thus was unable to emit in the NIR, while in strong alkaline conditions (pH ≥ 10), the reaction of CEs toward TTAP−AB was reduced [15,18], which hampered the release of fluorophore TTAP and prevented the generation of NIR fluorescence.Despite this, the TTAP−AB probe was still able to monitor CEs in physiological environments.

Sensing Mechanism
To confirm the reaction mechanism, the product of the reaction between TTAP−AB and CEs was separated and purified (Section S4), and its structure was analyzed using HRMS and 1 H NMR. As shown in Figure S2

Sensing Mechanism
To confirm the reaction mechanism, the product of the reaction between TTAP−AB and CEs was separated and purified (Section S4), and its structure was analyzed using HRMS and 1 H NMR. As shown in Figure S2   For a clearer understanding of the "turn-on" fluorescence response of the TTAP−AB probe to CEs, the fluorescence spectra of TTAP−AB and TTAP were studied in a DMSO-PBS mixture with different volume fractions of PBS buffer (f P ).As shown in Figure 4, TTAP−AB was almost non-emissive in pure DMSO, and the continuous addition of PBS buffer caused a negligible impact on fluorescence intensity.When f P reached levels of up to 99%, TTAP−AB formed nanoaggregates with an average size of 124 nm in the solution (Figure S3a), but the fluorescence intensity was hardly enhanced.Therefore, the TTAP−AB probe was shown to be non-emissive in both a dilute solution and an aggregate state, which might have been caused by the strong intramolecular charge transfer (ICT) effect from the triphenylamine-thiophene skeleton (strong electron donor) to the acrylonitrile-pyridinium moiety (strong electron donor), as well as the intense intermolecular dipole−dipole interaction [28,29].Meanwhile, the TTAP compound exhibited a weak pink emission in pure DMSO.Upon an increase in f P , the fluorescence intensity was slightly decreased because the PBS buffer improved the solvent polarity, which compelled the TTAP compound to form a twisted charge-separated conformation via intramolecular rotation, consequently weakening the fluorescence intensity [30].When f P reached levels of up to 70%, the fluorescence intensity at 692 nm was dramatically improved; when f P = 99%, the fluorescence intensity reached the maximum possible level, and the DLS results revealed that this solution formed nanoaggregates with an average size of 357 nm (Figure S3b).Therefore, the TTAP compound possessed AIE activity, and its aggregation was responsible for the TTAP-AB probe's NIR fluorescence response toward CEs.
Molecules 2024, 29, x FOR PEER REVIEW 7 of 15 (Figure S3a), but the fluorescence intensity was hardly enhanced.Therefore, the TTAP−AB probe was shown to be non-emissive in both a dilute solution and an aggregate state, which might have been caused by the strong intramolecular charge transfer (ICT) effect from the triphenylamine-thiophene skeleton (strong electron donor) to the acrylonitrile-pyridinium moiety (strong electron donor), as well as the intense intermolecular dipole−dipole interaction [28,29].Meanwhile, the TTAP compound exhibited a weak pink emission in pure DMSO.Upon an increase in fP, the fluorescence intensity was slightly decreased because the PBS buffer improved the solvent polarity, which compelled the TTAP compound to form a twisted charge-separated conformation via intramolecular rotation, consequently weakening the fluorescence intensity [30].When fP reached levels of up to 70%, the fluorescence intensity at 692 nm was dramatically improved; when fP = 99%, the fluorescence intensity reached the maximum possible level, and the DLS results revealed that this solution formed nanoaggregates with an average size of 357 nm (Figure S3b).Therefore, the TTAP compound possessed AIE activity, and its aggregation was responsible for the TTAP-AB probe's NIR fluorescence response toward CEs.

Cell Imaging of CEs
Inspired by the remarkable CE-sensitivity of the TTAP−AB probe in vitro, its application in living cells was then investigated.HepG2 cells were chosen as the model cell line due to their high CE expression [12].Prior to cell imaging, the cytotoxicity of TTAP−AB was evaluated using standard MTT assays.As shown in Figure S4, even if the concentration of TTAP−AB co-cultured with cells reached 25 µM, the cell survival rate was over 90%, indicating TTAP−AB's low cytotoxicity toward HepG2 cells and its suitability for the subsequent imaging experiments.Afterward, the sensing behavior of the TTAP−AB probe toward CEs in HepG2 cells was investigated.When the HepG2 cells were stained with only 5 µM TTAP−AB for 30 min, weak intracellular NIR fluorescence was observed (Figure 5a); when the cells were further incubated with different concentrations of CEs (0.10 U/mL, 0.15 U/mL, and 0.20 U/mL) for another 1 h, the emission intensities in the NIR channel showed a steady improvement with an increase in probe concentration (Figure 5b).Notably, the relative fluorescence intensity increased linearly with CEs levels from 0 to 0.20 U/mL (Figure S5), suggesting that TTAP−AB has promise for quantifying the levels of CEs in living cells.Thus, the TTAP−AB probe was able to image exogenous CEs in living cells.

Cell Imaging of CEs
Inspired by the remarkable CE-sensitivity of the TTAP−AB probe in vitro, its application in living cells was then investigated.HepG2 cells were chosen as the model cell line due to their high CE expression [12].Prior to cell imaging, the cytotoxicity of TTAP−AB was evaluated using standard MTT assays.As shown in Figure S4, even if the concentration of TTAP−AB co-cultured with cells reached 25 µM, the cell survival rate was over 90%, indicating TTAP−AB's low cytotoxicity toward HepG2 cells and its suitability for the subsequent imaging experiments.Afterward, the sensing behavior of the TTAP−AB probe toward CEs in HepG2 cells was investigated.When the HepG2 cells were stained with only 5 µM TTAP−AB for 30 min, weak intracellular NIR fluorescence was observed (Figure 5a); when the cells were further incubated with different concentrations of CEs (0.10 U/mL, 0.15 U/mL, and 0.20 U/mL) for another 1 h, the emission intensities in the NIR channel showed a steady improvement with an increase in probe concentration (Figure 5b).Notably, the relative fluorescence intensity increased linearly with CEs levels from 0 to 0.20 U/mL (Figure S5), suggesting that TTAP−AB has promise for quantifying the levels of CEs in living cells.Thus, the TTAP−AB probe was able to image exogenous CEs in living cells.
According to previous reports, CEs participate in the metabolism of many clinical drugs which also regulate their activity [1].Therefore, detecting CEs activity is extremely important for studying the relationship between CEs and drug metabolism, which will provide guidance for using drugs reasonably in therapies for related diseases.5-fluorouracil (5-FU), an antitumor drug, has been proven to up-regulate CEs activity in HepG2 cells [12,16].In view of this, the feasibility of using the TTAP−AB probe to monitor the activity of CEs regulated by 5-FU was investigated.As shown in Figure 6b, the untreated HepG2 cells had no background fluorescence, while intracellular NIR fluorescence could be clearly seen after co-incubation with TTAP−AB (5 µM) for 1 h (Figure 6e).Moreover, when HepG2 cells were successively treated with 5-FU (100 µM) and TTAP−AB (5 µM), the fluorescence intensity was obviously enhanced (Figures 6h and S6).However, when the cells were pre-incubated with 1 mM AEBSF (4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride, a CEs inhibitor) for 2 h, the fluorescence intensity in the NIR channel was significantly blocked (Figure 6k).These results show that the TTAP−AB probe could monitor the dynamic changes in CEs levels induced by the drug 5-FU and CEs inhibitors in living cells.According to previous reports, CEs participate in the metabolism of many clinical drugs which also regulate their activity [1].Therefore, detecting CEs activity is extremely important for studying the relationship between CEs and drug metabolism, which will provide guidance for using drugs reasonably in therapies for related diseases.5-fluorouracil (5-FU), an anti-tumor drug, has been proven to up-regulate CEs activity in HepG2 cells [12,16].In view of this, the feasibility of using the TTAP−AB probe to monitor the activity of CEs regulated by 5-FU was investigated.As shown in Figure 6b, the untreated HepG2 cells had no background fluorescence, while intracellular NIR fluorescence could be clearly seen after co-incubation with TTAP−AB (5 µМ) for 1 h (Figure 6e).Moreover, when HepG2 cells were successively treated with 5-FU (100 µM) and TTAP−AB (5 µМ), the fluorescence intensity was obviously enhanced (Figures 6h and S6).However, when the cells were pre-incubated with 1 mM AEBSF (4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride, a CEs inhibitor) for 2 h, the fluorescence intensity in the NIR channel was significantly blocked (Figure 6k).These results show that the TTAP−AB probe could monitor the dynamic changes in CEs levels induced by the drug 5-FU and CEs inhibitors in living cells.

Visualization of CEs Levels in Zebrafish
Encouraged by its remarkable cell imaging performance, the feasibility of using the TTAP−AB probe to visualize CEs in vivo was studied, using zebrafish larvae as a vertebrate model.As shown in Figure 7, the zebrafish exhibited weak NIR emissions in the abdomen after treatment with TTAP−AB (5 µM) only.This was mainly due to the normal

Visualization of CEs Levels in Zebrafish
Encouraged by its remarkable cell imaging performance, the feasibility of using the TTAP−AB probe to visualize CEs in vivo was studied, using zebrafish larvae as a vertebrate model.As shown in Figure 7, the zebrafish exhibited weak NIR emissions in the abdomen after treatment with TTAP−AB (5 µM) only.This was mainly due to the normal levels of CEs in zebrafish.When treated with different concentrations of 5-FU (50 µM, 100 µM) for 10 h, the probe-loaded zebrafish showed bright NIR fluorescence.In addition, the intensity of NIR fluorescence notably increased with increasing 5-FU concentration (Figure 7b,c), indicating that more endogenous CEs were being generated.However, after further incubation with AEBSF, the fluorescence signals decreased rapidly (Figure 7d), mainly due to the decomposition of the CEs.These results were consistent with those of cell imaging.These results indicate that the TTAP−AB probe was able to penetrate the tissues and monitor dynamic changes in CE levels in zebrafish.

Bioimaging of CEs in Tumor-Bearing Mice
As previously reported, CEs are mainly distributed in the liver, and their activity is closely related to cholesterol-induced liver injury and disease, especially liver cancer [12,19].In view of this, the ability of the TTAP−AB probe to image CEs in tumor-bearing mice was investigated.Before fluorescence imaging, the bio-safety of TTAP−AB was investigated in nude mice using histologic staining (hematoxylin and eosin, H&E) assays for 24 h.As shown in Figure S7, the TTAP−AB probe did not cause significant pathological changes in several primary organs, including the heart, liver, lung, kidney, and spleen; therefore, it showed no obvious toxicity in vivo and was considered suitable for imaging CEs in mice.Next, liver imaging was carried out.The mice in the control group were only injected with saline, and no obvious fluorescence signals were observed (Figure 8a), while in the experimental group, the mice were intravenously injected with TTAP−AB (100 µL, 200 µM), and the fluorescence signals in their abdomens increased quickly and reached the maximum levels at about 60 min, indicating that TTAP−AB was hydrolytically catalyzed by CEs.Simultaneously, the biodistribution of TTAP−AB was determined.The mice were euthanized, and their organs (heart, liver, spleen, lung, and kidney) were dissected for fluorescence imaging.As shown in Figure 8b, bright fluorescence signals were clearly observed in the liver, while there was almost no fluorescence in the other organs.These results indicated that CEs were mainly distributed in the liver, and the TTAP−AB probe was able to enter and accumulate in the livers of mice for imaging.
Afterward, imaging experiments were conducted on tumor-bearing mice.HepG2

Bioimaging of CEs in Tumor-Bearing Mice
As previously reported, CEs are mainly distributed in the liver, and their activity is closely related to cholesterol-induced liver injury and disease, especially liver cancer [12,19].In view of this, the ability of the TTAP−AB probe to image CEs in tumor-bearing mice was investigated.Before fluorescence imaging, the bio-safety of TTAP−AB was investigated in nude mice using histologic staining (hematoxylin and eosin, H&E) assays for 24 h.As shown in Figure S7, the TTAP−AB probe did not cause significant pathological changes in several primary organs, including the heart, liver, lung, kidney, and spleen; therefore, it showed no obvious toxicity in vivo and was considered suitable for imaging CEs in mice.Next, liver imaging was carried out.The mice in the control group were only injected with saline, and no obvious fluorescence signals were observed (Figure 8a

Materials and Instruments
Unless otherwise stated, all chemicals were purchased from commercial suppliers and used without further purification.Double-distilled water and chromatographic solvents were used for tests.A Bruker AV-400 spectrometer (Billerica, MA, USA) was employed to record 1 H NMR and 13 C NMR spectra.High-resolution mass spectra (HRMS) were obtained with a Thermo Scientific Q Exactive type mass spectrometer (Waltham, MA, USA).Fluorescence spectra and fluorescence quantum yield were collected using a Hitachi F-4500 fluorescence spectrometer (New Life Scientific, Cridersville, OH, USA).Dynamic light scattering (DLS) experiments were investigated with a ZEN3600 Malvern particle sizer (Counterpane Inc., Washington, DC, USA).Fluorescence images of living cells and zebrafish were obtained with a Zeiss LSM 880 confocal laser scanning microscope (Oberkochen, Germany).All fluorescence imaging of living mice was performed by a BLT AniView 600 small animal optical imaging system (Xi'an, China).

Synthesis of Fluorophore TTAP
Compound 2 was synthesized according to the previous method [31], and its synthetic procedure was exhibited in the Supporting Information (Section S5).After that, compound 2 (1.14 g, 3.00 mmol) and 4-pyridineacetonitrile (0.17 g, 3.60 mmol) were dissolved in 15 mL ethanol, and then 0.6 mL piperidine was added to the solution.This mixture was reacted at 80 a℃ for 8 h under a N2 atmosphere.After cooling to room temperature, the mixture was concentrated using rotary evaporators, and the remaining solid was purified by column chromatography (CH2Cl2/CH3OH as eluent, v/v = 30:1).The final product, TTAP, was obtained as a dark red solid (0.93 g, 64% yield). 1   Afterward, imaging experiments were conducted on tumor-bearing mice.HepG2 cells (human hepatocarcinoma cells) were implanted into BALB/c mice to induce the formation of liver tumors.As depicted in Figure 8c, when the tumor-bearing BALB/c mice were injected with the TTAP−AB probe (20 µL, 100 µM), the NIR signals of the tumor site showed a clear increase over time and reached the maximum intensity at 30 min, mainly due to the over-expression of CEs in liver tumors, successfully visualizing liver tumors in vivo.More remarkably, when the tumors were dissected, they still presented intense NIR fluorescence, but there were no fluorescent signals in the mice (Figure 8d), implying that TTAP−AB could assist in tumor imaging and resectioning.Overall, it is clear that the TTAP−AB probe is capable of achieving real-time imaging of CEs in liver tumors, exhibiting a great deal of potential for practical clinical applications.

Materials and Instruments
Unless otherwise stated, all chemicals were purchased from commercial suppliers and used without further purification.Double-distilled water and chromatographic solvents were used for fluorescence tests.A Bruker AV-400 spectrometer (Billerica, MA, USA) was employed to record 1 H NMR and 13 C NMR spectra.High-resolution mass spectra (HRMS) were obtained with a Thermo Scientific Q Exactive type mass spectrometer (Waltham, MA, USA).Fluorescence spectra and fluorescence quantum yield were collected using a Hitachi F-4500 fluorescence spectrometer (New Life Scientific, Cridersville, OH, USA).Dynamic light scattering (DLS) experiments were investigated with a ZEN3600 Malvern particle sizer (Counterpane Inc., Washington, DC, USA).Fluorescence images of living cells and zebrafish were obtained with a Zeiss LSM 880 confocal laser scanning microscope (Oberkochen, Germany).All fluorescence imaging of living mice was performed by a BLT AniView 600 small animal optical imaging system (Xi'an, China).

Synthesis of Fluorophore TTAP
Compound 2 was synthesized according to the previous method [31], and its synthetic procedure was exhibited in the Supporting Information (Section S5).After that, compound imaging, the HepG2 cells were divided into four groups.In the control group, HepG2 cells were only cultivated with PBS buffer for 30 min.In the second group, the cells were incubated with the TTAP−AB probe (5 µM) for 1 h.In the third group, HepG2 cells were pre-treated with the drug 5-fluorouracil (5-FU, 100 µM) for 6 h and then incubated with TTAP−AB (5 µM) for 1 h.In the final group, HepG2 cells were pre-treated with 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, a CEs inhibitor, 1 mM) for 2 h and then incubated with TTAP−AB (5 µM) for another 1 h.Prior to imaging, the above cells were washed with PBS buffer (10 mM, pH = 7.4) three times, and fluorescence imaging was performed using a confocal fluorescence microscope (λ ex = 488 nm; λ em = 650-750 nm).

Zebrafish Imaging
All zebrafish experiments were approved by the Experimental Animal Ethics Committee of Wuchang University of Technology (Approval Code: 20240415-063) and conducted according to the guidelines for animal experiments.Zebrafish embryos were purchased from Shanghai FishBio Co., Ltd.(Shanghai, China).Larval zebrafish (4 days old) were used for imaging, and they were divided into four groups.In a control group, zebrafish were only cultured with the TTAP−AB probe (5 µM) at 37 • C for 1 h.In the second group, zebrafish were grown with the 5-FU drug (50 µM) for 10 h and then stained with TTAP−AB (5 µM) at 37 • C for another 1 h.In the third group, zebrafish were stained with the 5-FU drug (100 µM) for 10 h and then incubated with TTAP−AB (5 µM) at 37 • C for another 1 h.In the last group, zebrafish were first treated with 5-FU (100 µM) for 10 h, then cultivated with AEBSF (1 mM) for 4 h, and finally stained with TTAP−AB (5 µM) at 37 • C for another 1 h.All zebrafish were washed three times with embryo media and then transferred to a confocal fluorescence microscope for imaging (λ ex = 488 nm; λ em = 650-750 nm).

Fluorescence Imaging in Mice
All animal experiments were approved by the Experimental Animal Ethics Committee of Wuchang University of Technology (Approval Code: 20240415-064) and conducted according to the guidelines for animal experiments.All BALB/c mice (18-20 g) were purchased from Shanghai Slac Laboratory Animal Co., Ltd.(Shanghai, China) and operated on in accordance with Wuchang University of Technology guidelines.
For histology and immunohistochemical staining, all BALB/c mouse tissues were immediately fixed in 10% formaldehyde after sacrifice.The histological examination was carried out according to conventional methods [12,13,19] via hematoxylin and eosin (H&E) staining.The morphology of any observed lesions was classified and recorded according to the classification criteria.
For imaging CEs in vivo, the mice were divided into two groups.In the control group, the mice were intravenously injected with 100 µL of PBS.In the experimental group, the mice were intravenously injected with the TTAP−AB probe (100 µL, 200 µM) for real-time recording.All the mice were anesthetized, and in vivo imaging was performed; next, they were used for the biodistribution studies.These mice were euthanized, and their organs (heart, liver, spleen, lung, and kidney) were dissected for fluorescence measurements.The fluorescence images were obtained using a BLT AniView 600 small animal optical imaging system (China) (λ ex = 600 nm, λ em = 650-750 nm).
To visualize CEs in tumor-bearing mice, HepG2 cells (5 × 10 7 cells) were subcutaneously injected into female BALB/c mice (18-20 g) to establish a mouse tumor model.After 20 days, the TTAP−AB probe (20 µL, 100 µM) was injected into the tumor-bearing mice.All the mice were anesthetized, and in vivo imaging was performed.Next, the liver tumors were removed from the mice after euthanasia.The fluorescence images were obtained using a BLT AniView 600 small animal optical imaging system (China) (λ ex = 600 nm, λ em = 650-750 nm).

Molecules 2024 , 15 Scheme 1 .
Scheme 1.The proposed mechanism of the TTAP−AB probe to detect CEs.

Scheme 2 .
Scheme 2. The synthetic route of the TTAP−AB probe.

Molecules 2024 , 15 Scheme 1 .
Scheme 1.The proposed mechanism of the TTAP−AB probe to detect CEs.

Scheme 2 .
Scheme 2. The synthetic route of the TTAP−AB probe.
, the HRMS spectrum displayed a major peak at m/z = 482.16848[M + H] + , indicating that the TTAP−AB probe was hydrolyzed by CEs to release the fluorophore TTAP.Moreover, according to the 1 H NMR spectra of TTAP−AB, TTAP, and the purified TTAP−AB+CEs product (Figure 3), the signals of the protons in the TTAP−AB acetoxy-benzyl recognition group at 5.79 ppm (H b ) and 2.27 ppm (H c ) both disappeared after reacting with CEs.Meanwhile, the signals of protons on the pyridine ring (H a ) in TTAP−AB showed a distinct upfield shift from 9.14 ppm to 8.15 ppm after the reaction.More importantly, the 1 H NMR spectra of the product from TTAP−AB+CEs were almost the same as those of TTAP.The above results demonstrate that the reaction of TTAP−AB with CEs produced the fluorophore TTAP.
, the HRMS spectrum displayed a major peak at m/z = 482.16848[M + H] + , indicating that the TTAP−AB probe was hydrolyzed by CEs to release the fluorophore TTAP.Moreover, according to the 1 H NMR spectra of TTAP−AB, TTAP, and the purified TTAP−AB+CEs product (Figure 3), the signals of the protons in the TTAP−AB acetoxy-benzyl recognition group at 5.79 ppm (Hb) and 2.27 ppm (Hc) both disappeared after reacting with CEs.Meanwhile, the signals of protons on the pyridine ring (Ha) in TTAP−AB showed a distinct upfield shift from 9.14 ppm to 8.15 ppm after the reaction.More importantly, the 1 H NMR spectra of the product from TTAP−AB+CEs were almost the same as those of TTAP.The above results demonstrate that the reaction of TTAP−AB with CEs produced the fluorophore TTAP.

Figure 3 . 1 H
Figure 3. 1 H NMR spectra of compound TTAP, the TTAP−AB probe, and the isolated product of TTAP−AB with CEs conducted in DMSO-d6.For a clearer understanding of the "turn-on" fluorescence response of the TTAP−AB probe to CEs, the fluorescence spectra of TTAP−AB and TTAP were studied in a DMSO-PBS mixture with different volume fractions of PBS buffer (fP).As shown in Figure 4, TTAP−AB was almost non-emissive in pure DMSO, and the continuous addition of PBS buffer caused a negligible impact on fluorescence intensity.When fP reached levels of up to 99%, TTAP−AB formed nanoaggregates with an average size of 124 nm in the solution

Figure 3 . 1 H
Figure 3. 1 H NMR spectra of compound TTAP, the TTAP−AB probe, and the isolated product of TTAP−AB with CEs conducted in DMSO-d 6 .

Figure 4 .
Figure 4.The fluorescence intensity of the TTAP−AB probe (15 µM) and compound TTAP (15 µM) in DMSO-PBS buffer mixture with different volume fractions of PBS buffer (fP %).Insert: the photos of TTAP in different solutions taken under 365 nm light.Error bars: standard deviation (SD), experimental times: n = 3.

Figure 4 .
Figure 4.The fluorescence intensity of the TTAP−AB probe (15 µM) and compound TTAP (15 µM) in DMSO-PBS buffer mixture with different volume fractions of PBS buffer (f P %).Insert: the photos of TTAP in different solutions taken under 365 nm light.Error bars: standard deviation (SD), experimental times: n = 3.
), while in the experimental group, the mice were intravenously injected with TTAP−AB (100 µL, 200 µM), and the fluorescence signals in their abdomens increased quickly and reached the maximum levels at about 60 min, indicating that TTAP−AB was hydrolytically catalyzed by CEs.Simultaneously, the biodistribution of TTAP−AB was determined.The mice were euthanized, and their organs (heart, liver, spleen, lung, and kidney) were dissected for fluorescence imaging.As shown in Figure 8b, bright fluorescence signals were clearly observed in the liver, while there was almost no fluorescence in the other organs.These results indicated that CEs were mainly distributed in the liver, and the TTAP−AB probe was able to enter and accumulate in the livers of mice for imaging.Molecules 2024, 29, x FOR PEER REVIEW 11 of 15TTAP−AB probe is capable of achieving real-time imaging of CEs in liver tumors, exhibiting a great deal of potential for practical clinical applications.